This project focuses on the stress-related peptide corticotropin-releasing factor (CRF), and the limbic-norepinephrine (NE) arousal system, as a target impacted by chronic alcohol exposure whose dysregulation may underlie, in part, the negative consequences of alcohol abuse. We base these studies on our previous evidence demonstrating a circuit designed to relay emotion-related information via the limbic CRF system to the locus coeruleus (LC)-NE arousal system. Dysregulation of the CRF receptor system in noradrenergic circuits following chronic alcohol use may play a critical role in abnormal responsiveness to stress and long-lasting vulnerability to relapse associated with exposure to stressful events. Monoaminergic nuclei, including the LC and dorsal raphe nucleus (DRN), are profoundly impacted by stress, and CRF transmission in these areas is implicated in stress-induced anxiety and reinstatement of substance abuse. Further, recent findings of sex differences in CRF receptor (CRFr) signaling and trafficking suggest a compromised ability to adapt to chronic stress in females that may have significant consequences under conditions of alcohol exposure; for example, although men display a higher prevalence for alcoholism, women exhibit more severe brain and other organ damage following binge or chronic alcohol abuse. The Aims of this fellowship will examine these issues in three ways. First the trafficking of CRFrs will be examined in male vs female rats following stress in chronic alcohol exposure conditions using high-resolution immunoelectron microscopy to test the hypothesis that CRFr trafficking is altered differentially between the sexes after chronic alcohol exposure. Next, in order to elucidate maladaptive changes in stress circuitry following alcohol exposure, c-Fos, combined with immunocytochemical detection of CRF, will be employed as a marker of neuronal activation to define the neurochemical phenotype of stress-activated limbic neurons in male vs female rats exposed to chronic alcohol, where we hypothesize that CRF will play a prominent role in neurons activated after stress. Finally, dual tract-tracing will be employed in combination with c-Fos and CRF immunohistochemistry to test the hypothesis that limbic afferents that are activated following stress prominently co-regulate brainstem noradrenergic and serotonergic nuclei. Elucidating potential sex differences in the mechanism of action of stress under alcohol exposure, and the role of limbic afferents in the coregulation of brainstem noradrenergic and serotonergic nuclei activated by stress, can advance our understanding of the pathophysiological basis of chronic alcohol abuse and serve as a guide for developing individualized therapies and novel targets for the treatment of alcohol dependence.